Abstract
Proteomic analysis of sensory organs such as the cochlea is challenging due to its small size and difficulties with membrane protein isolation. Mass spectrometry in conjunction with separation methods can provide a more comprehensive proteome, because of the ability to enrich protein samples, detect hydrophobic proteins, and identify low abundant proteins by reducing the proteome dynamic range. GELFrEE as well as different separation and digestion techniques were combined with FASP and nanoLC-MS/MS to obtain an in-depth proteome analysis of cochlear sensory epithelium from 30-day-old mice. Digestion with LysC/trypsin followed by SCX fractionation and multiple nanoLC-MS/MS analyses identified 3773 proteins with a 1% FDR. Of these, 694 protein IDs were in the plasmalemma. Protein IDs obtained by combining outcomes from GELFrEE/LysC/trypsin with GELFrEE/trypsin/trypsin generated 2779 proteins, of which 606 additional proteins were identified using the GELFrEE/LysC/trypsin approach. Combining results from the different techniques resulted in a total of 4620 IDs, including a number of previously unreported proteins. GO analyses showed high expression of binding and catalytic proteins as well as proteins associated with metabolism. The results show that the application of multiple techniques is needed to provide an exhaustive proteome of the cochlear sensory epithelium that includes many membrane proteins. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the data set identifier PXD000231.